The invention relates to the field of molecular biology and plant genetics. More specifically, this invention describes a technique to produce silk-like proteins plant expression systems.
Increasing demands for materials and fabrics that are both light-weight and flexible without compromising strength and durability has created a need for new fibers possessing higher tolerances for such properties as elasticity, denier, tensile strength and modulus. The search for a better fiber has led to the investigation of fibers produced in nature, some of which possess remarkable qualities. One of those fibers is silk, a group of externally spun fibrous protein secretions.
Silks are produced by over 30,000 species of spiders and by many other insects particularly in the order Lepidoptera (Foelix, R. F. (1992) Biology of Spiders, Cambridge, Mass. Harvard University Press). Few of these silks have been studied in detail. The cocoon silk of the domesticated silkworm Bombyx mori and the dragline silk of the orb-weaving spider Nephila clavipes are among the best characterized. Although the structural proteins from the cocoon silk and the dragline silk are quite different from each other in their primary amino acid sequences, they share remarkable similarities in many aspects. They are extremely glycine and alanine-rich proteins. Fibroin, a structural protein of the cocoon silk, contains 42.9% glycine and 30% alanine. Spidroin 1, a major component of the dragline silk, contains 37.1% glycine and 21.1% alanine. They are also highly repetitive proteins. The conserved crystalline domains in the heavy chain of the Fibroin and a stretch of polyalanine in Spidroin 1, are repeated numerous times throughout entire molecules. These crystalline domains are surrounded by larger non-repetitive amorphous domains in every 1 to 2 kilobases in the heavy chain of Fibroin, and by shorter repeated GXG amorphous domains in tandem in Spidroin 1. They are also shear sensitive due to their high copy number of the crystalline domains. During fiber spinning, the crystalline repeats are able to form anti-parallel xcex2-pleated sheets, so that silk protein is turned into semi-crystalline fiber with amorphous flexible chains reinforced by strong and stiff crystals (Kaplan et al., (1997) in Protein-Based Materials, McGrath, K., and Kaplan, D. Eds, Birkhauser, Boston, pp 104-131).
Traditional silk production from silkworm involves growing mulberry leaves, raising silkworm, harvesting cocoons, and processing of silk fibers. It is labor intensive and time consuming and therefore prohibitively expensive. The natural defects of the silkworm silk, such as the tendency to wrinkle and the irregularity of fiber diameter further limits its application. Similarly, the mass production of the dragline silk from spiders is not plausible because only small amounts are available from each spider. Furthermore, multiple forms of spider silks are produced simultaneously by any given spider. The resulting mixture has less application than a single isolated silk because the different spider silk proteins have different properties and are not easily separated. Thus, the prospect of producing commercial quantities of spider silk from a natural source is not a practical one and there remains a need for an alternate mode of production.
By using molecular recombination techniques, one can introduce foreign genes or artificially synthesized DNA fragments into different host organisms for the purpose of expressing desired protein products in commercially useful quantities. Such methods usually involve joining appropriate fragments of DNA to a vector molecule, which is then introduced into a recipient organism by transformation. Transformants are selected using a selectable marker on the vector, or by a genetic or biochemical screen to identify the cloned fragment.
While the techniques of foreign gene expression in the host cell are well known in the art and widely practiced, the synthesis of fiber forming foreign polypeptides containing high numbers of repeating units poses unique problems. Genes encoding proteins of this type are prone to genetic instability due to the repeating sequences which result in truncated product instead of the full size protein.
In spite of the above mentioned difficulties, the expression of fiber forming proteins is known in the art. Ferrari et al. (U.S. Pat. No. 5,770,697) disclose methods and compositions for the production of polypeptides having repetitive oligomeric units such as those found in silk-like proteins (SLPs) and elastin-like proteins by the synthetic structural genes. The DNA sequences of Ferrari encode peptides containing an oligopeptide repeating units which contains at least 3 different amino acids and a total of 4-30 amino acids, there being at least 2 repeating units in the peptide and at least 2 identical amino acids in each repeating unit.
The cloning and expression of silk proteins of B. mori are also known. Ohshima et al. (Proc, Natl. Acad. Sci. USA, 74, 5363 (1977)) reported the cloning of the silk Fibroin gene complete with flanking sequences of B. mori into E. coli. Petty-Saphon et al. (EP 320702) disclose the recombinant production of silk Fibroin and silk Sericin from a variety of host including E. coli, Sacchromyces cerevisiae, Pseudomonas sp., Rhodopseudomonas sp., Bacillus sp., and Strepomyces sp. 
Progress has also been made in the cloning and expression of spider silk proteins. Xu et al. (Proc, Natl. Acad. Sci. USA, 87, 7120 (1990)) report the determination of the sequence for a portion of the repetitive sequence of a dragline like protein, Spidroin 1, from the spider Nephila clavipes, based on a partial cDNA clone.
Lewis et al. (EP 452925) disclose the expression of spider silk proteins (Spidroin 1 and 2) including protein fragment and variants, of Nephila clavipes from transformed E. coli. 
Lombardi et al. (U.S. Pat. No. 5,245,012) teach the production of recombinant spider silk protein comprising an amorphous domain or subunit a crystalline domain or subunit where the domain or subunit refers to a portion of the protein containing a repeating amino acid sequence that provides a particular mechanostructural property.
The recent advances in cDNA sequencing of cocoon silk and dragline silk have permitted the synthesis of artificial genes for silk-like proteins (SLPs) with sequence and structural similarity to the native proteins. These artificial genes mimicked sequence arrays of natural cocoon silk from B. mori and dragline silk from N. clavipes, and had been introduced into microorganisms such as Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. SLPs had been produced in these microorganisms through fermentation [Cappello, J., Crissman, J. W. (1990) Polymer Preprints 31:193-194; Cappello et al., (1990) Biotechnol. Prog. 6:198-202; Fahnestock and Irwin, Appl. Microbiol. Biotechnol. (1997), 47(1), 23-32; Prince et al, (1995) Biochemistry 34:10879-10885; Fahnestock and Bedzyk, 1997, Appl. Microbiol. Biotechnol. (1997), 47(1), 33-39 and commonly owned WO 9429450].
Plants are becoming a favorite host for foreign gene expression. Many recombinant proteins have been produced in transgenic plants (Franken et al., Curr. Opin. Biotechnol. 8:411-416, (1997); Whitelam et al., Biotechnol. Genet. Eng. Rev. 11:1-29, (1993). Plant genetic engineering combines modem molecular recombination technology and agricultural crop production. Although a variety of silk-like and fiber forming proteins have been expressed in microbial systems, similar expression systems have not been developed in plants. Zhang et al. teach the expression of an elastin-based protein polymer in transgenic tobacco plants (Zhang et al., Plant Cell Rep. (1996), 16(3-4), 174-179). Although this represents the expression of a repetitive sequence in plants, the elastin polypeptide bears little resemblance to silk-like peptides and thus the feasibility of SLP expression in plants can not be predicted based on this work.
To date, there are no reported examples of recombinant silk or SLP production in plants. One possible explanation for this lies in the the striking compositional and structural differences between Silks and SLP""s and native plant proteins. For example, SLP proteins are very glycine and alanine-rich, highly repetitive, and semi-crystalline in structure. These are characteristics not found in most plant proteins. Thus, introduction and expression of SLP genes in plant cells may pose a number of difficulties. For example, the repetitive sequence of SLP gene may be a target for DNA deletion and rearrangement in plant cells.
Alternatively, translation of glycine and alanine-rich SLP might prematurely exhaust glycine and alanine and tRNAs pools in plant cells. Finally, accumulation of semicrystalline SLP may be recognized and degraded by the house-keeping mechanisms in the plant.
The methods recited above for the expression of silk and SLP are useful for production in microbial systems, however fail to teach the production of silk or SLP in plants. The use of a plant platform for the production of silk and silk-like proteins has several advantages over a microbial platform. For example, as a renewable resource, a plant platform requires far less energy and materiel consumption than microbial methods. Similarly, a plant platform represents a far greater available biomass for protein production than a microbial system. Finally, the fact that silks are natural proteins suggests production of high levels of silk will not be toxic to the host.
The problem to be solved, therefore is to provide a method to produce synthetic silk or SLP in commercially useful quantities at relatively low cost.
Applicants have solved the stated problem by providing a method to express and produce silk or SLP using plant expression systems.
The present invention provides a method for the production of silk-like proteins in a green plant comprising:
a) providing a green plant containing a SLP expression cassette having the following structure:
P-SLP-T
xe2x80x83wherein:
P is a promoter suitable for driving the expression of a silk-like protein gene;
SLP is a transgene encoding a mature silk-like protein; and
T is a 5xe2x80x2 terminator;
xe2x80x83wherein each of P, SLP and T are operably linked such that expression of the cassette results in translation of the silk-like protein;
b) growing said green plant under conditions whereby said transgene is expressed and the silk-like protein is produced; and
c) optionally recovering said silk-like protein.
Additionally the invention provides plants comprising an expression cassette expressing a silk-like protein derived from the silks produced by Bombyx mori and Nephila clavipes. Specifically the silks and silk-like proteins of the present invention may be natural or variants and will have the general formula:
[(A)n-(E)q-(S)q-(X)p-(E)q-(S)q]i
wherein:
A or E are different non-crystalline soft segments of about 10 to 25 amino acids having at least 55% Gly;
S is a semi-crystalline segment of about 6 to 12 amino acids having at least 33% Ala, and 50% Gly;
X is a crystalline hard segment of about 6-12 amino acids having at least 33% Ala, and 50% Gly; and
xe2x80x83wherein,
n=2, 4, 8, 16, 32, 64, or 128;
q=0, 1, 2, 4, 8, 16, 32, 64, or 128;
p=2, 4, 8, 16, 32, 64, or 128;
i=1-128; and
where p greater than n or q.